【作者】 袁凯；

【导师】 姚雪彪；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2009， 博士

【摘要】 在整个细胞周期中,有丝分裂期是最为壮观同时也最具潜在威胁的一个阶段。有丝分裂中每一个编排的错误都可能导致非整倍体的产生以及基因组不稳定性,并最终引起细胞死亡或者肿瘤发生。因此,研究有丝分裂进程中染色体运动可塑性以及动态性的有机协调过程就显得尤为重要。借助于生物光子学以及化学生物学研究手段,我探讨了真核生物有丝分裂过程中染色体准确分离的分子调控机制。有丝分裂过程中染色体的分离是由纺锤体微管与着丝粒之间动态的相互作用所介导的,而蛋白激酶与磷酸酶的信号级联通路在其中起着掌控作用。有大量证据表明蛋白激酶PLK1在有丝分裂进入时被激活,并在有丝分裂各个阶段发挥着不可缺少的作用。然而,我们对PLK1激酶活性在进入有丝分裂后的时空调节的分子机制,以及PLK1在有丝分裂过程中的动态可塑性等方面的了解还很不全面。我们建立了基于荧光共振能量转移的PLK1活性光学探针,以期描绘PLK1激酶活性在整个有丝分裂期的时空动力学特征。我的实验结果显示在有丝分裂的前期及前中期动点上的PLK1激酶活性较高,随着中期染色体双极定向的建立,PLK1的活性开始下降。PLK1活性的下降与动点之间张力的建立成存在时间序列性。利用针对有丝分裂激酶的小分子抑制剂,我的研究进一步揭示了Aurora B在维持与协调动点上PLK1激酶活性过程中的作用。为此,我的研究首次揭示了Aurora B-PLK1激酶调节轴在动点张力及染色体动力学调控中的潜在机制。在对PLK1在有丝分裂期的动态可塑性的进一步研究中,我们发现PLK1与蛋白磷酸酶hCdc14A之间存在相互作用。通过体外实验,我们揭示hCdc14A的磷酸酶活性受其分子内相互作用的抑制性调节。而PLK1通过结合并磷酸化修饰hCdc14A,部分解离了hCdc14A的分子内相互作用从而起到激活hCdc14A磷酸酶活性的作用。更重要的是,过量表达hCdc14A的模拟磷酸化突变体引起染色体排列异常以及有丝分裂前中期的阻断。这一现象暗示着PLK1对hCdc14A的动态调节在协调有丝分裂各个事件序列性中至关重要。在细胞分裂过程中,染色体的准确分裂直接关乎父代的基因组能否忠实可靠的传递到子代细胞中。在有丝分裂后期,当securin被anaphase-promoting complex降解之后,由separase介导的对染色体之间cohesion的不可逆的切割最终实现了染色体的分离。然而,之前的报道认为separase在有丝分裂期定位于中心体,这就使得在有丝分裂起始时separase如何有效的对姐妹染色体之间的cohesion进行切割成为一个问题。利用一种新的separase抗体,我们发现separase在有丝分裂期的表达量增加,并在有丝分裂末期被降解。利用该抗体进行免疫荧光染色时,我们惊奇的发现从有丝分裂前期至中期separase都分布在染色体上,在有丝分裂后期染色体分离之后separase的染色体定位随即消失。对分离出来的染色体进行染色更是发现separase在着丝粒处有富集现象。为了进一步探讨介导separase染色体定位的机制以及有丝分裂激酶在其中可能的调控作用,我们利用RNAi抑制了几种有丝分裂激酶的表达,结果发现separase的染色体定位可能受到Aurora B的调控。同时,在经过针对Aurora B激酶活性的小分子抑制剂hesperadin处理的细胞中,separase的染色体定位也几乎消失。这些结果暗示Aurora B的激酶活性在协调separase染色体定位及姐妹染色单体分离的过程中发挥着作用。作为着丝粒处cohesion的保护神,Sgol协同PP2A一起防止了姐妹染色单体在有丝分裂后期起始之前的提前分离。另外它在感受动点之间张力以及维持动点一微管连接的稳定性等方面的功能也屡有报道。在体外Sgol能被anaphase-promotingcomplex泛素化。然而调控Sgol在有丝分裂中期向后期转变时从动点上解离下来的机制目前还不清楚。我们的研究发现Sgol在有丝分裂过程中被蛋白酶水解,随后我们证明了在体外Sgol是钙依赖的蛋白水解酶m-calpain的底物。进一步在细胞水平的实验表明,人为升高有丝分裂期细胞内钙离子浓度导致Sgol从动点上解离,而如果在此过程中利用E64d抑制calpain的活性则能维持Sgol的动点定位。结合之前报道的钙离子浓度在有丝分裂后期起始时升高,我们认为,m-calpain通过水解Sgol参与了有丝分裂中期向后期转变的过程。总而言之,在本文中我们探讨了在有丝分裂期PLK1激酶活性的时空调节及其本身的动态可塑性;揭示了separase的染色体定位并对其机制进行了解析;提出了mcalpain水解Sgol这一调控有丝分裂中期向后期转变的新机制。这些成果为我们明确了未来研究的方向,并对我们全面了解有丝分裂过程中染色体分离的分子机制提供了帮助。更多还原

【Abstract】 Mitosis is the most dramatic,as well as critical event in the cell cycle.Errors in the choreography of mitotic chromosome movements can lead to aneuploidy or genetic instability,fostering cell death or tumor development.Therefore it is essential to delineate how cellular plasticity and dynamics are achieved and orchestrated during mitotic progression.Combined with biophotonic and chemical biological approaches, here I attempted to delineate the molecular mechanisms underlying faithful chromosome segregation in mammalian cell division.Chromosome segregation in mitosis is orchestrated by dynamic interactions between spindle microtubules and kinetochores,which in turn are governed by protein kinase-and phosphatase-signaling cascades.Growing evidences indicate that Polo-like kinase 1 （PLK1） is activated during mitotic entry and plays an essential role in mitosis from start to finish.However,the molecular illustration of spatiotemporal dynamics of PLK1 in mitosis is unavailable.To illuminate of PLK1 activity in space and time,I have developed a fluorescence resonance energy transfer（FRET）-based optical sensor to report PLK kinase activity of kinetochore.Using this optical sensor,my study shows that kinetochore-centered PLK1 activity is high in prophase and prometaphase,and drops upon the achievement of chromosome bi-orientation in metaphase.Importantly,our analysis demonstrates that PLK1 kinase dynamics provides an accurate readout of interkinetochore tension.Using chemical modulators combined with real-time imaging the PLK1 sensor,my studies reveal that PLK1 kinase gradient at kinetochore is governed by Aurora B.Our findings suggest a novel regulatory mechanism by which Aurora B and PLK1 kinases cooperate to ensure accurate kinetochore-microtubule attachment and chromosome plasticity in mitosis.Our molecular delineation of PLK1 kinase function in mitosis led to the identification of a novel interaction between PLK1 and hCdc14A phosphoatase.Our biochemical characterization reveals that hCdc14A phosphatase activity latent in early mitosis via its intra-molecular association.PLK1 interacts with and phosphorylates hCdc14A,therefore stimulates hCdc14A’s phosphatase activity by partially releasing its auto-inhibition. Importantly,overexpression of the phospho-mimicking mutant caused aberrant chromosome alignment with a prometaphase delay,suggesting the temporal regulation of hCdc14A activity by PLK1 is critical for orchestrating mitotic events.Accurate segregation of chromosomes,initiated by abrupt and irreversible dissolution of sister-chromatid cohesion at anaphase,is crucial for the faithful inheritance of parental genomes during cell division.The dissolution of sister-chromatid cohesion is catalyzed by separase after the destruction of securin by the anaphase-promoting complex. However,separase was localized to the mitotic centrosome,raising the question as how separase hydrolyzes sister-chromatid cohesion of centromere at the anaphase onset.Using a panel of separase antibodies,we found that separase protein is accumulated in mitosis and degraded at the end of telophase.Surprisingly,in our immunofluorescence microscopic analyses,separase was found to be associated with mitotic chromosomes from prophase to metaphase and dissociated from chromosomes in anaphase right after sister chromatids separation.Staining of isolated mitotic chromosomes from nocodazole-arrested cells revealed that separase is concentrated at the centromeric cohesion.To examine if any mitotic kinases are responsible for chromosomal localization of separase, we carried out RNAi-mediated knockdown and found the association of separase with mitotic chromosomes is a function of Aurora B.Consistent with the phenotype seen in the Aurora B-repressed cells,inhibition of Aurora B kinase by hesperadin prevents the association of separase with chromosomes.Our results suggested that Aurora B kinase activity helps coordinate the association of separase with chromosomes and the initiation of sister-chromatid separation.It has been postulated that accurate kinetochore-microtubule attachment and tension across the sister kinetochore initiates anaphase events.As the guardian spirit of centromeric cohesion,Sgol collaborates with PP2A to orchestrate the temporal order of sister chromatide separation at the anaphase onset.Temporal control of Sgo1 degradation allows a timely resolution of sister chromatid cohesion by separase.However,the mechanism regulating its displacement from kinetochore at metaphase-anaphase transition remains unclear.Our in vitro cleavage results suggested Sgo1 is a novel substrate of the Ca²⁺-dependcnt protease m-calpain.The physiological relevance of calpain-Sgo1 interaction was demonstrated by the liberation of Sgo1 from kinetochore upon elevation of intracellular calcium.Importantly,the Sgo1 dissociation from kinetochore in response to the intracellular calcium rise was blocked by calpain inhibitor E64d,demonstrating the role of calpain-mediated proteolysis in dissociation of Sgo1 from kinetochore.Our findings illustrate a novel regulatory mechanism by which spatiotemporal dynamics of calcium spike in mitosis cooperate with chromosome segregation machinery to ensure the chromosome plasticity in mitosis.In sum,my studies illustrate the spatiotemporal dynamics and plasticity of PLK1 activity in mitosis.In addition,my analyses reveal that Aurora B and PLK1 kinases cooperate to ensure accurate kinetochore-microtubule attachment in mitosis.Together with the discovery calpain-Sgo1 inter-relationship established,my studies shed new light on the mitotic orchestration underlying chromosome segregation.更多还原